Nucleic acid probes and methods for detecting Candida glabrata DNA in blood

ABSTRACT

Provided is an isolated double-stranded nucleic acid consisting essentially of the nucleotide sequences defined in the Sequence Listing by SEQ ID NOs: 5-9. These are the ITS2 sequences for C. albicans, C. parapsilosis, C. tropicalis, C. glabrata and C. krusei. A method of diagnosing systemic candidiasis in a subject is also provided. The method comprises the steps of: (a) collecting blood from the subject into tubes containing detergent, polypropylene glycol, sodium poyantholesulfonate, and sodium ethylene diamine tetraacetic acid; (b) lysing Candida cells using ZYMOLYASE®-100T with agitation; (c) extracting and precipitating the DNA from the lysed cells; (d) amplifying the precipitated DNA using universal fungal primer pairs derived from the internal transcribed spacer regions of the Candida ribosomal DNA; and (e) detecting amplified DNA from Candida by hybridizing the amplified DNA with a probe that selectively hybridizes with Candida DNA, the presence of amplified DNA indicating systemic candidiasis.

This application is a division of application Ser. No. 08/065,845, filedMay 20, 1993 which status is now U.S. Pat. No. 5,426,027.

BACKGROUND OF THE INVENTION

Candida albicans is a commensal of the gastrointestinal tract. C.albicans, and to a lesser extent several other related species, are ofincreasing importance as opportunistic pathogens in immunocompromisedhosts. A dimorphic, diploid yeast with no known sexual cycle, C.albicans is an endogenous organism that can be isolated from skin andmucosal tissues of persons whose immune systems are intact. However,perturbations of the immune or endocrine systems can createopportunities for Candida species to convert from a commensal state toinvade tissues either locally or systemically. An example of thisopportunism is the oral-esophageal or vaginal candidiasis that isencountered in association with HIV infection. In C. albicans, thenuclear rDNA genes encoding the 5S, 18S, 5.8S, and 28S rRNAs are foundas 50-100 copy tandem repeats of approximately 10 kb unit length onchromosome seven (Magee et al., 1987, Thrash-Bingham and Gorman, 1992).The 5S rDNA gene (121 bp) is flanked by two nontranscribed regionslocated between the small and large subunits, and collectively termedthe intergenic spacer (IGS). Ribosomal 5.8S sequences have been compiledfrom a variety of eukaryotes (Dams et al., 1988). In addition, sequenceanalysis of the 5.8/28S internally transcribed spacer (ITS) region hasshown strain variation within at least one fungal species (O'Donnell,1992), while other species have demonstrated complete conservation(Mitchell et al., 1992). Strain-specific restriction polymorphisms(RFLPs) have previously been observed in the IGS region for C. albicans(Magee et al., 1987).

An opportunistic fungus, C. albicans also causes systemic disease inseverely immunocompromised hosts. It is the most causative species ofdisseminated candidiasis followed by C. tropicalis, C. parapsilosis, andC. glabrata (Odds, 1988). Dissemination occurs when Candida is spreadvia the bloodstream or by invasion of mucosal surfaces to internalorgans (Odds, 1988). High-risk patient populations include individualswith malignancy or neutropenia, those receiving chemotherapy and/ormultiple antibiotics, and those with indwelling catheters or low birthweight infants (Armstrong, 1989).

Diagnosis of systemic candidiasis is complicated by the absence ofclinically distinguishing signs, frequently negative blood cultures, andthe absence of a reliable serological test to detect infection.Currently, disseminated candidiasis is often diagnosed by a minimum ofat least two positive blood cultures (Odds, 1988). However, bloodculture alone is clearly not sufficient for the diagnosis ofdisseminated candidiasis since as many as 50% of disseminatedcandidiasis cases are diagnosed at autopsy (Telenti, et al. 1989). Thenephrotoxicity of amphotericin B, the drug of choice forimmunocompromised patients with disseminated disease, precludes its usefor prophylaxis.

These facts, in conjunction with the difficulty of reliably culturingCandida from the blood and the lack of a sensitive and specificserological test to detect disease, underscore the need to developalternative diagnostic approaches.

Technology has been developed for the detection of bacterial and viralDNA from the bloodstream of infected patients through the use of thepolymerase chain reaction (PCR). The PCR amplifies genomic DNAgeometrically so that it may be detected by agarose gel electrophoresis,Southern blotting, or dot blot hybridization (Miyakawa et al. 1992,Kafatos et al. 1979, Lasker et al. 1992).

PCR-based diagnostic methods may provide increased sensitivity relativeto blood culture techniques since viable organisms are not required foramplification or detection. There has only been one report to datedescribing the detection of C. albicans cells in infected patient bloodthrough the use of PCR-amplified DNA (Buckman et al. 1990). Buchman etal. lysed C. albicans cells with ZYMOLYASE and proteinase K andextracted the DNA with phenol and chloroform. The limit of sensitivityby this method was 120 cells per ml of whole blood. As described, thismethod was time consuming, labor-intensive, repeatedly used toxicchemicals (phenol and chloroform), and has not been shown to be readilyreproducible. In addition, a single copy gene, the cytochrome P-450gene, was the target for DNA amplification, thus making the method muchless sensitive. Miyakawa et al. described improved sensitivity by use ofSouthern blot hybridization for the detection of PCR products fromCandida DNA (Miyakawa et al. 1991). The limit of sensitivity by Southernblot in their study was 10 cells per ml of urine and did not addressdetection in blood.

The ability to detect Candida in blood is crucial for the rapid andaccurate diagnosis of systemic candidiasis, because detection from urineor mucosal secretions can be confused with the normal commensal statusof the organism or a localized non-disseminated infections. The presentinvention provides a rapid method for the isolation, release,purification and amplification of C. albicans DNA from blood and otherbody fluids of infected patients. This method minimizes the use ofphenol and chloroform and uses universal fungal primers to themulti-copy ITS region of rDNA, to enhance detection of Candida DNA. Theinvention provides a rapid approach to species identification throughthe use of non-conserved regions of the ITS2 flanked by highlyconserved, functional domains.

SUMMARY OF THE INVENTION

The present invention provides an isolated double-stranded nucleic acidconsisting essentially of the nucleotide sequence defined in theSequence Listing by SEQ ID NO:5. This is the C. albicans ITS2 sequenceand includes a nucleic acid comprising a nucleotide sequence that isspecific for C. albicans. Further examples of an isolated doublestranded nucleic acid of the present invention consist essentially ofthe nucleotide sequences defined in the Sequence Listing by SEQ ID Nos:6-9. These are the ITS2 sequences for C. parapsilosis, C. tropicalis, C.glabrata and C. krusei. These nucleic acids can include a nucleotidesequence that is specific for the respective organism.

An isolated nucleic acid that specifically hybridizes with orselectively amplifies a nucleic acid of the invention or fragmentsthereof is also contemplated. An isolated nucleic acid complementary tothe above nucleic acid is also provided.

A method of diagnosing systemic candidiasis in a subject is alsoprovided. The method comprises the steps of: (a) collecting blood fromthe subject into tubes containing detergent, polypropylene glycol,sodium polyanetholesulfonate, and sodium ethylene diamine tetraaceticacid; (b) lysing Candida cells using ZYMOLYASE®-100T with agitation; (c)extracting and precipitating the DNA from the lysed cells; (d)amplifying the precipitated DNA using universal fungal primer pairsderived from the internal transcribed spacer regions of the Candidaribosomal DNA; and (e) detecting amplified DNA from Candida byhybridizing the amplified DNA with a probe that selectively hybridizeswith Candida DNA, the presence of amplified DNA indicating systemiccandidiasis.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides an isolated double-stranded nucleic acidconsisting essentially of the nucleotide sequence defined in theSequence Listing by SEQ ID NO: 5. This includes the C. albicans ITS2sequence. By "isolated" is meant separated from other nucleic acidsfound in the naturally occurring organism. The nucleic acid comprises anucleotide sequence that is specific for C. albicans. By "specific" ismeant a sequence which does not hybridize with other nucleic acids toprevent determination of an adequate positive hybridization with nucleicacids from C. albicans. Probes which "specifically hybridize" with thedouble-stranded nucleic acid are hybridizing with one of the two strandswhen in single stranded form.

A further example of an isolated double stranded nucleic acid of thepresent invention consists essentially of the nucleotide sequencedefined in the Sequence Listing by SEQ ID NO: 6. This includes the ITS2sequence for C. parapsilosis. This nucleic acid comprises a nucleotidesequence that is specific for C. parasilosis.

Another example of the isolated double stranded nucleic acid of theinvention consists essentially of the nucleotide sequence defined in theSequence Listing by SEQ ID NO: 7. This includes the C. tropicalis ITS2sequence. This nucleic acid comprises a nucleotide sequence that isspecific for C. tropicalis.

A still further example of the isolated double stranded nucleic acid ofthe invention consists essentially of the nucleotide sequence defined inthe Sequence Listing by SEQ ID NO: 8. This includes the C. glabrata ITS2sequence. This nucleic acid comprises a nucleotide sequence that isspecific for C. glabrata.

Another example of the isolated double stranded nucleic acid of theinvention consists essentially of the nucleotide sequence defined in theSequence Listing by SEQ ID NO: 9. This includes the C. krusei ITS2sequence. This nucleic acid comprises a nucleotide sequence that isspecific for C. krusei.

An isolated nucleic acid that specifically hybridizes with orselectively amplifies a nucleic acid of the invention or fragmentsthereof is also contemplated. An isolated nucleic acid complementary tothe above nucleic acid is also provided. The sequences can be selectedbased on the nucleotide sequence and the utility of the particularsequence. More specifically the invention provides isolated nucleicacids that specifically hybridize with the nucleic acids consistingessentially of the nucleotide sequences defined in the Sequence Listingby SEQ ID NOs: 5-9.

The term "consisting essentially of", as used herein includesmodifications to the nucleic acids of the invention as long as thespecificity (genus or species) of the nucleic acids is maintained.Likewise, fragments used as primers or probes can have substitutions solong as enough complementary bases exist for specific hybridization(Kunkel et al. Methods Enzymol. 1987: 154:367, 1987).

The nucleic acid can have homology with nucleotide sequences present inmore than one Candida species. Such a nucleic acid sequence shared withother Candida species can be used, for example, as a primer tosimultaneously amplify nucleic acids from more than one Candida species.The amplified nucleic acids can then be detected using the specificnucleic acids described herein to permit either genus specific orspecies specific diagnosis. Thus, the specific nucleic acid can bespecific for the genus Candida and can be used to detect any candidiasisin methods such as polymerase chain reaction, ligase chain reaction andhybridization.

A method of diagnosing systemic candidiasis in a subject is alsoprovided. The method comprises the steps of: (a) collecting blood fromthe subject into tubes containing detergent, polypropylene glycol,sodium polyanetholesulfonate, and disodium ethylene diamine tetraaceticacid ((Na)₂ EDTA); (b) lysing Candida cells using zymolase-100T withagitation; (c) extracting and precipitating the DNA from the lysedcells; (d) amplifying the precipitated DNA using universal fungal primerpairs derived from the internal transcribed spacer regions of theCandida ribosomal DNA; and (e) detecting amplified DNA from Candida byhybridizing the amplified DNA with a probe that selectively hybridizeswith Candida DNA, the presence of amplified DNA indicating systemiccandidiasis.

In the method, the lysis step can use the lysis buffer from theISOQUICK® kit in addition to ZYMOLASE®-100T. The agitation step can beby rocking at about 16 cycles per minute. The extracting step can usethe extraction matrix in the ISOQUICK® kit. In the amplification step ofthe above method, one of the primers of the primer pair is derived fromthe internal transcribed spacer 1 (ITS1) and the other primer of theprimer pair is derived from the internal transcribed spacer 2 (ITS2).Alternatively, one of the primers of the primer pair is derived from theinternal transcribed spacer 3 (ITS3) and the other primer of the primerpair is derived from the internal transcribed spacer 4 (ITS4). Thedetecting step hybridization can be by dot blot hybridization using agenus or species specific Candida probe.

In the method of detecting systemic candidiasis, the DNA that isamplified can be from C. albicans and the probe can specificallyhybridize with a specific nucleotide sequence of the nucleic acid of SEQID NO: 5 as described in Example 2. By using the other specific nucleicacids as provided herein, the method of Example 2 can be used to detectany of the other Candida species as taught herein. If the DNA that isamplified is from C. parapsilosis, the probe specifically hybridizeswith a specific nucleotide sequence of the nucleic acid of SEQ ID NO: 6.If the DNA that is amplified is from C. tropicalis, the probespecifically hybridizes with a specific nucleotide sequence of thenucleic acid of SEQ ID NO: 7. If the DNA that is amplified is from C.glabrata, the probe specifically hybridizes with a specific nucleotidesequence of the nucleic acid of SEQ ID NO: 8. If the DNA that isamplified is from C. krusei, the probe specifically hybridizes with aspecific nucleotide sequence of the nucleic acid of SEQ ID NO: 9. Anucleic acid having homology with more than one Candida species can alsobe used as a probe that specifically hybridizes with Candida DNA todetect systemic candidiasis.

Additionally, it is contemplated that the nucleic acids (e.g., probesand primers) can be attached to or labeled with (covalently ornon-covalently) a detectable moiety. The probes may be suitably labeledusing, for example, a radio label, enzyme label, fluorescent label,biotin-avidin label and the like for subsequent visualization in theexample of the dot blot hybridization procedure taught in Example 2. Anexample of such a labeled nucleic acid is the digoxigenin-UTP labelledprobe provided in Example 2, although others can be readily generatedusing standard methods (See, e.g., Sambrook et al., 1989). The nucleicacids specific for a given Candida species can each be labeled with adistinct detectable moiety, such that species specific probes forseveral species can be used with the same sample of amplified DNA topermit species specific diagnosis. The distinct label for each speciesspecific probe can be detected in the sample if DNA from the particularspecies is present in the subject.

The detection of fungal DNA as described herein can also be performedusing a ligase chain reaction (LCR). Essentially, this reaction, knownto those of skill in the art, involves the use of, for each region to bedetected, two primers that hybridize to the same strand of the targetDNA, either abutting each other or with one or two nucleotides betweenthe two primer sequences (i.e., "immediately 5'" or "immediately 3'" tothe junction). The ligase reaction is performed, and the products areelectrophoresed through a gel that can detect very small fragments, suchas an SDS-polyacrylamide gel. A positive result is one in which aproduct equal in size to the sum of the two primers is produced, as thisindicates the presence of all of the target DNA region. It is preferablethat three reactions be run in three separate tubes, targeted atdetecting (1) the first Junction, (2) the second junction and (3) aninternal sequence as a positive LCR control. If one wants toelectrophorese all LCR products together through the gel, primers mustbe carefully chosen such that their individual sizes can bedistinguished from the predicted size of any LCR products.Alternatively, the product of each reaction can be electrophoresedseparately. Primers are preferably exactly homologous to the targetregion and of a size between approximately 20-40 nucleotides.

The following examples are intended to illustrate, but not limit, theinvention. While they are typical of those that might be used, otherprocedures known to those skilled in the art may be alternativelyemployed.

EXAMPLES Example 1 Nucleotide Sequence Analysis of the ITS2 Region ofCandida albicans and Related Species

Yeast strains and maintenance

All Candida isolates have been previously characterized by assimilation(API) profiles and morphology (Van der Walt and Yarrow, 1984). Inaddition, all C. albicans and C. parapsilosis isolates have previouslybeen electrophoretically karyotyped and are known to represent distinct,non-related strains (Lasker et al., 1989). All isolates were grown andmaintained on yeast-peptone-dextrose (YPD) medium (Guthrie and Fink,1991). For DNA extractions, 10 ml of overnight cultures grown on YPD at37° C. were washed twice in 1×TE buffer and the DNA extracted bystandard procedures (Sambrook et al., 1989). Prior to PCRamplifications, DNA was digested with EcoRI restriction endonuclease(New England Biolabs), electrophoresed on 1.0% agarose gels, and stainedwith ethidium bromide (EtBr) to verify concentration and purity.

PCR amplification and DNA sequencing

Taq polymerase, buffers, and conditions for PCR were those supplied bythe vendor (Perkin-Elmer/Cetus), using 100 ng genomic DNA per reaction.For primary amplifications, 35 cycles of 95° C., 55° C., and 72° C. atone min. intervals were followed by a five min. final extension at 72°C. The following "universal" ITS primers were used, for which calculatedTm's have previously been reported (White et al., 1990):

ITS1 5' TCC GTA GGT GAA CCT GCG G 3' (SEQ ID NO: 1)

ITS3 5' GCA TCG ATG AAG AAC GCA GC 3' (SEQ ID NO: 2)

ITS4 5' TCC TCC GCT TAT TGA TAT GC 3' (SEQ ID NO: 3)

Primer ITS1 is to a conserved 3' domain in the 18S nuclear subunit.Primer ITS3 is approximately 25 bp from the end of the 5.8S subunit, andITS4 is a reverse primer to a conserved region of the nuclear largerDNA. In addition, a -21M13 forward primer sequence (Messing et al.1981) was added at the 5' end to primers ITS1 and ITS4 for sequencing inthe forward and reverse directions, respectively, and consisted of thesequence: 5' GTA AAA CGA CGG CCA G 3' (SEQ ID NO: 10) where the terminal5' T of ITS1 and ITS4 made 17 bp of the 18 bp annealing sequence. Frompreliminary experiments it was determined that the addition of thissequence did not alter the nature of the derived PCR product. Theaqueous phase of the primary PCR reaction was ethanol-precipitated,dried, and resuspended in 8 μl TE buffer. The entire amount was loadedinto single wells of a 1.5% agarose, 1.0% NuSieve agarose gel (Lehmannet al. 1992), electrophoresed at 110 V., and stained with EtBr. Single,intensely staining bands of the appropriate size were excised and theDNA was extracted in Spin-X cellulose acetate columns (Costar, Inc.) for30 min. at 40° C., 13000×g. The DNA was then ethanol-precipitated,washed twice in 70% ET/OH, dried briefly, and resuspended in H₂ O forsequencing. Automated DNA sequencing (Smith et al. 1986), was performedusing the Applied Biosystems Catalyst 800 workstation, with the "Prism"dye-primer dideoxy-sequencing reactions (Sanger et al. 1977), usingconditions supplied by the vendor (Applied Biosystems). The precipitatedDNA was dried and resuspended in 6 μl of formamide/50 mM EDTA (5:1),denatured for 2 min. at 90° C. and loaded on an Applied Biosystems model373A DNA sequencer. All DNAs were sequenced in both forward and reverseorientations, and multiple runs were performed for all species and moststrains within a given species.

5.8s rDNA

5.8S sequence alignments were performed both manually and with the"pileup" program from the University of Wisconsin Genetics ComputerGroup (GCG) package (Devereux et al., 1984). ITS alignments wereperformed in all possible pairwise combinations using the Needleman andWunsch algorithm as implemented by GCG (Needleman and Wunsch 1970). DNAparsimony and bootstrap analysis was performed using the "Phylip"programs of Felsenstein (Felsenstein 1982), implemented on a micro-vax(Digital Equip. Corp.) cluster. Dendrograms were constructed using theglobal option and using a variety of different species as the outgroup(Felsenstein 1985). Other 5.8S sequences were: Neurospora crassa,Schizosaccharomyces pombe, Saccharomyces cerevisiae, Pneumocystiscarinii, Fusarium sambucium, Epichloe typhina, Cephalosporiumacremonium, Lentinula edodes.

For C. albicans and C. parapsilosis, where multiple strains wereanalyzed, there was complete nucleotide conservation within the entire159 bp 5.8S region. The greatest degree of diversity for the speciesused in this study was found in the two relatively unconserved regionsbetween bp 79-85 and bp 118-136. The overall average degree of diversitybetween the Candida species was approximately three percent. The minimumdegree of diversity was found between C. tropicalis and C. parapsilosis,with a single C-A transversion at bp 62. Interestingly, both C. albicansand C. krusei contained A-G transitions in the termination consensusTCATTT.

A phylogenetic analysis was performed with all known fungal 5.8Ssequences using strict parsimony as implemented by Felsenstein andstatistical bootstrap analysis (Felsenstein 1982; 1985). P. carinii wasused as the outgroup considering previous findings based on 18S analysisusing a larger database of eukaryotic organisms (Edman et al. 1988).There were a total of 47 informative sites for the number of fungalsequences compiled, including 4 single base pair gaps. Re-analysis ofthe data set without gaps did not significantly alter the tree topology.The cumulative number of positive selections out of 100 total iterationsis given for each branch point. The derived tree does not differsignificantly from previous research using a weighted differencealgorithm for 18S sequences, and supports the view that these speciesare related such that C. albicans, C. parapsilosis and C. tropicalis aremore closely aligned than C. krusei within a clade. Likewise, C.glabrata appears more distantly related and can equally be placed at anumber of positions within the larger branch of yeast-like fungi. It isgenerally accepted that values of 70 or greater out of 100 randomlytested samples will represent similar trees to a significant degree ofprobability.

ITS2 rDNA

The sequences of the ITS2 regions for C. albicans, C. parapsilosis, C.tropicalis, C. glabrata and C. krusei are shown in the Sequence Listingas SEQ ID NOs: 5-9.

A total of ten C. albicans isolates, representing typical andmorphologically (or physiologically) atypical strains, were found to beidentical at the nucleotide level within the ITS region. Similarly, fivestrains of C. parapsilosis, displaying a wide range of electrophoretickaryotypes and randomly amplified polymorphisms (RAPD), were alsoidentical to the type strain for the species. The entire length of theITS region was found to be species specific.

Similar to the results of the 5.8S alignments, we found that C.albicans, C. parapsilosis, and C. tropicalis were also most homologousin this ITS region. This homology extended for the first 57 bp 5'immediately adjacent to the termination of the 5.8S sequence. Incontrast, the 3' region displayed little homology. For C. krusei and C.glabrata there was no apparent homology either to each other or tomembers of the C. albicans group over this entire ITS region. Sequenceswere aligned in all possible pairwise combinations (Needleman and Wunsch1970), and the average degree of similarity was found to beapproximately 40 percent.

Analysis of the ITS2 region has revealed that C. albicans, and possiblyother closely related species, displays no interstrain variation. Inthis respect this species resembles the opportunistic fungusCryptococcus neoformans, and is unlike the plant pathogen Fusariumsambucinum which displays variation in this region.

Example 2 Detection of DNA from Candida albicans Cells in Blood by Useof the Polymerase Chain Reaction (PCR)

Growth of C. albicans

C. albicans strain 36B was grown on Sabouraud's dextrose agar Emmonsslants for 48 h at 25° C. Cells were harvested by washing each slantwith 5 ml of 0.85% NaCl, centrifuged at 1500×g for 10 min, andresuspended to the appropriate concentration in freshly collectedrabbit's blood or 0.85% saline.

Yeast cell lysis and DNA purification

Blood from adult female rabbits (New Zealand White, Myrtle's RabbitFarm) was collected from the central ear artery into ISOLATOR 10®microbial tubes (Wampole Laboratories, Cranbury, N.J.) containing anaqueous solution of 1 unit of purified saponin, 8 ml/L polypropyleneglycol, 9,6 g/L Na polyanetholesulfonate and 16 g/L (Na)₂ EDTA;EDTA-coated tubes (Becton Dickinson, Rutherford, N.J.); or heparinizedtubes (Becton Dickinson). C. albicans strain 36B (Quebec GynecologicalInstitute, Montreal, Quebec) cells were then introduced and samples werecentrifuged at 3000×g for 30 min. Supernatants were removed and an equalvolume of deionized water was added to lyse residual blood cells.Remaining C. albicans cells were washed in 0.85% NaCl and pelleted bycentrifugation at 1500×g for 10 min. ISOLATOR 10® tubes have provensuperior to other blood collection systems for the recovery of viable C.albicans cells from blood (Jones, 1990). The use of the ISOLATOR 10®tubes for blood collection resulted in PCR amplification of candidal DNAwhereas the use of EDTA- or heparin-coated tubes did not.

C. albicans DNA was extracted and purified using the ISOQUICK® nucleicacid extraction kit according to the manufacturer's instructions withthe addition of ZYMOLASE®-100T, to allow its use with fungi, since theISOQUICK® kit was developed by MicroProbe Corporation for the isolationand purification of DNA from only mammalian cells and gram negativebacteria. Briefly, pelleted cells were suspended in 100 μl of samplebuffer for 15 min after which 100 μl of lysis buffer was added. Themixture was incubated at 25° C. for 1 h. Selected samples containedZYMOLYASE (ZYMOLYASE®-100T, Seikagaku Corp., Tokyo, Japan; 5 mg/ml in1.0M sorbitol, 0.1M trisodium citrate, and 0.1% 2-mercaptoethanol)during the lysis step and were rocked at 16 cycles per min to optimizebreakage of C. albicans cells. The addition of ZYMOLYASE®-100T to thelysis step allowed for successful adaptation of the ISOQUICK® kit foruse with C. albicans cells. Alternatively, C. albicans cells weredisrupted using a mini bead beater (Biospec Products, Bartlesville,Okla.) (Glee et al. 1987). Cells (1 ml) were delivered into Sarstedtmicrofuge tubes containing 1 ml of 0.5 mm diameter glass beads andbeaten at maximum speed for 2 min. A third method released C. albicansDNA by boiling 1×10⁷ cells per ml in 2 mls of deionized water in anEppendorf microcentrifuge tube for 30 min. Mechanical disruption of C.albicans cells by bead beating or boiling was less effective inproducing PCR amplifiable DNA; these methods may be too harsh, resultingin shearing or fragmentation of DNA. For precipitation of the DNA sodiumacetate and other components of the ISOQUICK® kit were used as directed.

After lysis, DNA was purified with the extraction matrix provided in theISOQUICK® kit, precipitated with sodium acetate in the presence ofisopropanol, and the precipitated DNA was dried by vacuum centrifugationfor 15 min.

PCR amplification of genomic DNA

Universal fungal primer pairs, ITS1 and 2 or ITS3 and 4, synthesized bythe CDC core facility, and the GeneAmpR DNA amplification reagent kitusing native Taq DNA polymerase (250 U, Perkin Elmer Cetus, Alameda,Calif.) were used for PCR amplification of genomic DNA (Saiki et al.1988). These primers amplify DNA from all fungi and some parasites.Examples of the ITS1, ITS2, ITS3 and ITS4 primers are shown in theSequence Listing as SEQ ID NOs: 1, 4, 2 and 3, respectively. Thereaction consisted of the following: 53.5 μl of double distilled,sterile water, 10 μl of 10X reaction buffer, 16 μl of a mixture ofequimolar (1.25 mM) quantities of dATP, dCTP, dGTP, and dTTP, 5 μl of 20μM ITS1 or 3, 5 μl of 20 μM ITS2 or 4, 10 μl of target DNA, 0.5 μl ofTaq polymerase, and 6 μl of 25 mM MgCl₂. Samples were overlaid withmineral oil prior to placement in the thermal cycler (Perkin ElmerCetus) to minimize evaporation during DNA amplification. Samples wereinitially denatured in the thermal cycler at 95° C. for 5 min. This wasfollowed by 30 cycles of: denaturation at 95° C. for 1 min, annealing at50° C. for 2 min, and extension at 72° C. for 1.5 min. Final extensionoccurred at 72° C. for 5 min.

After amplification, mineral oil was discarded. An equal volume ofchloroform was added to the samples which were then centrifuged for 5min at 4100×g to extract residual mineral oil. The aqueous layer wasremoved and the DNA precipitated from it by adding 2 volumes of ice-cold100% ethanol followed by incubation for 30 min at -70°C. Samples werethen centrifuged for 1 min at 4100×g, the ethanol removed, the samplesdried under vacuum, and resuspended in 20 μl of TE buffer (20mM Trisplus 1 mM EDTA, pH 8.0). Amplified DNA was visualized after agarose (1%agarose plus 1% Nu-Sieve in TE buffer) gel electrophoresis by ethidiumbromide staining or by dot blot hybridization analysis.

Dot blot hybridization

C. albicans strain 3307 DNA was used as a probe for the dot blot. Tomake the probe, 20 ng of C. albicans 3307 genomic DNA was PCR-amplifiedusing ITS1 and ITS2 or ITS3 and ITS4 as primer pairs. The PCR productwas then electrophoresed on an agarose gel and the resultant DNA bandcut out of the gel. The product was extracted from the gel by thefreeze-squeeze method of Thuring et al (Thuring et al., 1975). The DNAprobe was labeled by incubating overnight with digoxigenin-dUTP from anonradioactive-DNA labeling and detection kit according to themanufacturers instructions ("Genius" kit, Boehringer Mannheim,Indianapolis, Ind.). Other genus or species specific probes derived fromthe nucleic acids of SEQ ID NOs: 5-9 can also be used in this method.

Samples were prepared for the dot blot (Kafatos et al., 1979, Lasker etal., 1992) by diluting 10 μl of C. albicans DNA to 25 μl with TE buffer,adding NaOH to a final concentration of 0.3M, and incubating for 10 minat 25° C. An equal volume of 2.0M ammonium acetate was then added toeach sample on ice. Each sample was then dotted under vacuum onto anitrocellulose filter using a dot blot apparatus (BioRad, Richmond,Calif.) according to the manufacturer's instructions. The filter wasthen removed from the apparatus and dried at 80° C. under vacuum for 2h. The dried filter was placed in a plastic bag, sealed, andprehybridized with single-stranded salmon sperm DNA (10 μg/ml) overnightin a 65° C. water bath.

The digoxigenin-labeled probe was denatured by boiling for 5 min, addedto the filter in the plastic bag, and placed in a 65° C. water bathovernight. The filter was then washed twice for 30 min each in citratedsaline (0.3M NaCl with 0.03M sodium citrate, pH 7.0) and 0.1% SDS at 60°C. (Lasker et al., 1992). Washed filters were incubated for 30 min at25° C. with an anti-digoxigenin antibody (1:5000) labeled with alkalinephosphatase. Chromogen (nitroblue tetrazolium salt and5-bromo-4-chloro-3-indolyl phosphate) was added (Lasker et al., 1992)and color developed for 6 h at 25° C. in the dark.

"Booster" PCR amplification

"Booster" PCR amplification was performed by the method of Ruano et al.(Ruano et al., 1989). Briefly, the same protocol as outlined above wasused, but after 15 cycles of PCR amplification, samples were removedfrom the thermal cycler and fresh primers were added to a finalconcentration of 40 μM. The samples were then returned to the thermalcycler for 15 additional cycles and final extension. The level ofsensitivity of detection of the PCR product from cells introduced intoblood was improved from 10⁵ cells per ml to 10³ cells per ml as detectedby ethidium bromide stained agarose gels. However, the specificity ofthis system was poor since the negative control became positive.

Detection of PCR amplified products from C. albicans in saline byagarose gel electrophoresis

A comparison of C. albicans DNA isolated and purified from saline usingthe ISOQUICK® kit alone to that obtained by the use of ZYMOLYASE®-100Tplus the kit was performed. C. albicans cells (10^(7/) ml saline) werelysed at either 37° C. or 25° C. The combined use of ZYMOLYASE®-100T andthe ISOQUICK® kit (at either 25° C. or 37° C.) resulted in enhancedrecovery of purified DNA relative to the kit alone.

To determine the sensitivity of the ZYMOLASE®-100 T plus ISOQUICK®method for cell breakage and DNA purification, C. albicans cells werethen serially diluted in saline (10⁷ to 10¹ cells per ml) beforebreakage. Ethidium bromide stained agarose gels demonstrated that 10³cells per ml could be detected by this method. Based on these results,all subsequent experiments used ZYMOLYASE®-100T followed by DNApurification with the ISOQUICK® kit at 25° C.

Detection of PCR amplified products of C. albicans in blood by agarosegel electrophoresis

To determine if the ZYMOLYASE®-100T plus ISOQUICK® kit method could beused to detect C. albicans in blood, 10⁷ C. albicans cells per ml wasintroduced into freshly collected rabbit's blood as described above.Blood was collected into one of the following: ISOLATOR 10® microbialtubes, EDTA-coated tubes, or heparinized tubes. Amplified DNA wasdetected in the samples prepared from cells introduced into blood drawninto ISOLATOR 10® tubes only. No DNA was detected in samples where bloodhad been drawn into either only EDTA- or only heparin-coated tubes.

The sensitivity of detection for C. albicans DNA in blood using theZYMOLYASE®-100T plus ISOQUICK® kit method was determined by seriallydiluting C. albicans cells (10⁷ to 10¹ cells per ml) in blood drawn intoISOLATOR 10® tubes. Using agarose gel electrophoresis and ethidiumbromide staining, 10⁵ cell per ml could be detected.

Dot blot hybridization for detection of PCR amplified products of C.albicans in blood or saline.

In an effort to improve the sensitivity for detection of C. albicansDNA, a comparison was performed of the ethidium bromide-stained agarosegel method to a dot blot hybridization method for the detection of thePCR product. The dot blot method allowed detection of 10¹ cells per mlin saline versus 10³ cells per ml detected by agarose gelelectrophoresis and ethidium bromide staining. The sensitivity fordetection of the PCR product of C. albicans cells introduced into bloodwas 10¹ cells per ml by the dot blot method versus 10⁵ cells per ml forethidium bromide stained agarose gels detection. The probe used for theabove dot blot was C. albicans-specific. C. tropicalis DNA and humanplacental DNA did not react in the dot blot, supporting the specificityof the probe. Thus, the methods taught herein are capable of detectingCandida DNA in clinical samples such as blood.

Universal fungal primers as described herein provide the potential foramplification of DNA from all fungi. However, by using a C.albicans-specific DNA probe, as in the above-described dot blothybridization step, the test was specific for C. albicans. The dot blotassay can be conducted using specific probes for other Candida species,as described herein, or other fungi. Furthermore, because the presentmethod can gently extract DNA from clinical samples, the method can alsouse vital, bacterial or other fungal primers for the PCR reactionfollowed by specific DNA probes for each genus or species in the dotblot as described above.

Throughout this application various publications are referenced withinparentheses. Full citations for these publications may be found at theend of the specification immediately preceding the Sequence Listing. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

REFERENCES

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Barns, S. M., Lane, D. J., Sogin M. L., Bibeau, C. and Weisburg, W. G.(1991) Evolutionary relationships among pathogenic Candida species andrelatives. J. Bacteriol. 173: 2250-2255.

Buchman, T. G., M. Rosser, W. G. Merz, and P. Charache. 1990. Detectionof surgical pathogens by in vitro DNA amplification. Part I. Rapididentification of Candida albicans by in vitro amplification of afungus-specific gene. Surgery 108: 338-347.

Dams, E., Hendriks, L., Van de Peer, Y., Neefs, J. and Smits, G. (1988)Compilation of small ribosomal subunit RNA sequences. Nucl. Acids Res.16: r87-r174.

Devereux, J., Haeberli, P. and Smithies, O. (1984) A comprehensive setof sequence analysis programs for the VAX. Nucl. Acids Res. 12: 387-397.

Edman, J. C., Kovacs, J. A., Masur, H., Santi, D. V., Elwood, H. J. andSogin, M. L. (1988) Ribosomal RNA shows Pneumocystis carinii to be amember of the fungi. Nature (London). 334: 519-522.

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Felsenstein, J. (1985) Confidence limits on phylogenies: an approachusing the bootstrap. Evolution. 39: 783-791.

Glee, P. M., P. J. Russell, J. A. Welsch, J. C. Pratt, and J. E. Cutler.1987. Methods of DNA extraction from Candida albicans. Anal. Biochem.164: 207-213.

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Jones, J. M. 1990. Laboratory diagnosis of invasive candidiasis. Clin.Microbiol. Rev. 3: 32-45.

Kafatos, F. C., C. W. Jones, and A. Efstraliadis. 1979. Determination ofnucleic acid sequence homologies and relative concentrations by a dotblot hybridization procedure. Nucl. Acids Res. 3: 1541-1552.

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Lasker, B. A., Carle, G. F., Kobayashi, G. S. and Medoff, G. (1989)Comparison of the separation of Candida albians chromosome-sized DNA bypulsed-field gel electrophoresis techniques. Nucl. Acids Res, 17:3783-3793.

Lehmann, P. F., Lin, D. and Lasker, B. A. (1992) Genotypicidentification and characterization of species and strains within thegenus Candida by using random amplified polymorphic DNA. J. Clin. Micro,30: 3249-3254.

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    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 10                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       TCCGTAGGTGAACCTGCGG19                                                         (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       GCATCGATGAAGAACGCAGC20                                                        (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       TCCTCCGCTTATTGATATGC20                                                        (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GCTGCGTTCTTCATCGATGC20                                                        (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 151 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       CTCCCTCAAACCGCTGGGTTTGGTGTTGAGCAATACGACTTGGGTTTGCTTGAAAGACGG60                TAGTGGTAAGGCGGGATCGCTTTGACAATGGCTTAGGTCTAACCAAAAACATTGCTTGCG120               GCGGTAACGTCCACCACGTATATCTTCAAAC151                                            (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 124 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       CTCCCTCAAACCCTCGGGTTTGGTGTTGAGCGATACGCTGGGTTTGCTTGAAAGAAAGGC60                GGAGTATAAACTAATGGATAGGTTTTTTCCACTCATTGGTACAAACTCCAAAACTTCTTC120               CAAA124                                                                       (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 141 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       CTCCCTCAAACCCCCGGGTTTGGTGTTGAGCAATACGCTAGGTTTGTTTGAAAGAATTTA60                ACCGTGGAAACTTATTTTAAGCGACTTAGGTTTATCCAAAACGCTTATTTTGCTAGTGGC120               CACCACAATTTATTTCATAAC141                                                      (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 231 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       CCTTCTCAAACACATTGTGNTTGGTANTGAGTGATACNCNNTTTTGATNTAACTTNAAAT60                TGTAGGCCATATCAGTATGTGGGACACGAGNGCAAGCTTCTCTATTAATCTGCTGCTGCT120               TTGCGCGAGCGGCGGGGGTTAATACTCTATTAGGTTTTACCAACTCGGTGTTGATCTAGG180               GAGGGATAAGTGAGTGTTTTGTGCGTGCTGGGCAGACAGACGTCTTTAAGT231                        (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 177 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       GAGCGTCGTTTCCATCTTGCGCGTGCGCAGAGTTGGGTGAGCGGANGTACCGACGTGTAA60                AGAGCGTCGGAGCTGCGACTCNNCTGAAAGGGAGCNNANTGGCCCGAGCGAACTAGACTT120               TTTTTNAGGGNCCGTTTTGGGCCCCAGAACGNAGTTTTNCCNAGGNCAACAAAAAGN177                  (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      GTAAAACGACGGCCAG16                                                            __________________________________________________________________________

What is claimed is:
 1. An isolated double stranded nucleic acidconsisting of the nucleotide sequence defined in the Sequence Listing bySEQ ID NO:
 8. 2. An isolated nucleic acid of up to 231 nucleotides thatspecifically hybridizes with the nucleic acid of claim
 1. 3. A method ofdiagnosing systemic candidiasis in a subject comprising the steps of:a)collecting blood from the subject into tubes containing detergent,polypropylene glycol, sodium polyanetholesulfonate, and sodium ethylenediamine tetraacetic acid; b) lysing Candida cells using ZYMOLASE®-100Twith agitation; c) extracting and precipitating the DNA from the lysedcells; d) amplifying the precipitated DNA using universal fungal primerpairs that amplify the internal transcribed spacer regions of theCandida ribosomal DNA; and e) detecting amplified DNA from as few as 1Candida glabrata cell per 100 microliters of blood by hybridizing theamplified DNA with a probe that specifically hybridizes with the nucleicacid of claim 1, the presence of hybridization indicating systemiccandidiasis.
 4. The method of claim 3, wherein the lysis step furtheruses the lysis buffer from the ISOQUICK® kit.
 5. The method of claim 3,wherein the agitation is by rocking at 16 cycles per minute.
 6. Themethod of claim 3, wherein the extracting step uses the extractionmatrix in the ISOQUICK® kit.
 7. The method of claim 3, wherein one ofthe primers of the primer pair is derived from the internal transcribedspacer 1 and the other primer of the primer pair is derived from theinternal transcribed spacer
 2. 8. The method of claim 3, wherein one ofthe primers of the primer pair is derived from the internal transcribedspacer 3 and the other primer of the primer pair is derived from theinternal transcribed spacer
 4. 9. The method of claim 3, wherein thedetecting step hybridization is by dot blot hybridization.